CD24 Inhibits The Liver Fibrosis In Mouse Cholestasis Model By Regulating The Ly6C^lo Macrophage

Objectives:Liver fibrosis occurs in most types of chronic liver diseases.Advanced liver fibrosis results in cirrhosis,liver failure,and portal hypertension and often requires liver transplantation.Hepatic macrophages and stellate cells play central roles in the processes of liver fibrosis.During liver damage,macrophage is activated and secrets fibrogenic cytokines,leading to the fibroblast-like transformation of liver stellate cell.CD24 is reported to closely relate to the development and activation of many immune cells,and it is a negative regulator for inflammatory response.In this study,we aimed to investigate the regulation of CD24 for hepatic macrophage subsets and the molecular mechanisms of BDL-induced liver fibrosis.Methods:Wild-type（WT）and CD24 knockout(CD24^-/-)mice were used to establish bile duct ligation（BDL）and sham operation（SHAM）models and were sacrificed on the7th day after modeling.Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of ALT and AST to evaluate the degree of liver injury.Liver tissue sections were stained with Sirius red（Sirius）and immune fluorescent staining（IF）ofα-SMA to evaluate liver fibrosis status.Real-time PCR was used to detect the expression of fibrosis-related genes Acta2,Col1a1 and Mmp13 in liver tissue.IF staining of liver tissue sections with specific anti-F4/80 and anti-CD24 fluorescent antibodies was used to detect the number of macrophages and the overall expression of CD24 molecules in liver tissue after modeling.The non-parenchymal cells in liver tissue were isolated for flow cytometry（FCM）analysis to determine the proportion of macrophages and the expression of CD24 in liver.Subsequently,non-parenchymal cells from the liver of WT and CD24^-/-mice treated with BDL were isolated,and CD11b^hiF4/80^intLy6C^lo macrophages(Ly6C^lomacrophages)were obtained by flow sorting（FACS）.Extract the RNA of Ly6c^lomacrophages,evaluate the purity and integrity of RNA and use it for RNA-seq.After comparing the RNA-seq result from the reference genome,the quantitative expression information on the gene is obtained.Then the statistical analysis of gene quantitative expression information,genes with significantly different expression levels were screened;GO function enrichment analysis,KEGG pathway analysis,and protein interaction network analysis were performed on the differential gene sets.Subsequent experiments are based on the result of RNA-seq.Firstly,the non-parenchymal cells in the liver of WT and CD24^-/-mice with BDL were isolated for sorting the CD11b^intF4/80^hi(F4/80^hi macrophages),the CD11b^hiF4/80^intLy6C^hi(Ly6C^himacrophages)and the Ly6C^lo macrophages.Then real-time PCR was used to detect the expression of fibrosis-related cytokines Tgfb1,Pdgfa,Il10,Il6,and Vegfa in the three macrophage subpopulations.In addition,after obtaining the sorted macrophages,the macrophages were co-culture with stellate cells to analyze the effects of different macrophage groups on activation of stellate cells.Result:Compared with the SHAM mice,the degree of injury and fibrosis in BDL-induced mice was increased,and the number of macrophages and the expression of CD24 in liver tissue were also increased.Besides,the expression of CD24 on Ly6C^lomacrophages was also up-regulated in BDL mice（p<0.001）.CD24^-/-mice showed more severe liver tissue damages and fibrosis compared with WT mice.And the total number of Ly6C^lo macrophages was increased in BDL model（p<0.01）.The previous results suggest that CD24 may affect the BDL-induced liver fibrosis mainly by regulating the Ly6C^lo macrophages.Here,the Ly6C^lo macrophages were isolated from the liver for RNA-seq analysis.After KEGG gene enrichment analysis,the Focal adhesion,Cytokine-cytokine receptor interaction,PI3K-Akt signaling pathway,and MAPK signaling pathway showed statistical significance.These pathways are all related to the PDGF family,which plays an important role in fibrosis generation.The RNA-seq results also showed that PDGF family-related genes,such as Pdgfa,Pdgfb,and Pdgfd were up-regulated in the Ly6C^lo macrophage of CD24^-/-mice compared with WT mice.However,other cytokines related to macrophage-induced fibrosis,such as TGF-β,VEGF and IL6 related genes,are not significantly upregulated in CD24^-/-Ly6C^lo macrophage.To explore the underlying mechanisms of CD24 regulating liver fibrosis mediated by hepatic macrophages,we sorted the subsets of macrophages from WT and CD24^-/-mice treated with BDL,Real-time PCR detection showed that the mRNA expression of Pdgfa and Tgfb1 is up-regulated in Ly6C^lo macrophages from CD24^-/-mice,consistent with the RNA-seq analysis.Finally,the co-culture experiments of macrophages with stellate cells revealed a major contribution to HSCs activation by the Ly6C^lo subset macrophage in CD24^-/-mice.Conclusion:In the mouse BDL model,CD24 may play an important role in inhibiting the activation of stellate cells by regulating hepatic Ly6C^lo macrophages,thereby suppressing the progression of liver fibrosis.